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K Marcoe In Cell User Ge Meeting  2008
 

K Marcoe In Cell User Ge Meeting 2008

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Screening for Mechanisms of Hepatotoxicity: Phospholipidosis, Steatosis, Apoptosis and Inflammatory Markers

Screening for Mechanisms of Hepatotoxicity: Phospholipidosis, Steatosis, Apoptosis and Inflammatory Markers

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    K Marcoe In Cell User Ge Meeting  2008 K Marcoe In Cell User Ge Meeting 2008 Presentation Transcript

    • Screening for Mechanisms of Hepatotoxicity: Phospholipidosis, Steatosis, Apoptosis and Inflammatory Markers IN Cell User Meeting May 14, 2008 Karen Marcoe MDS Pharma Services
    • Drug-Induce Hepatotoxicity Liver major site of metabolism for most drugs Based on safety, hepatotoxicity recognized as a leading cause for drug withdrawal Toxicity of new drug candidates routinely evaluated just prior to compounds moving into clinical trial Late stage In vivo toxicity studies have problems − Costly (multiple animal species requirements) − Large amounts of compounds − Significant investment of resources tied to late findings In vitro early stage toxicity studies afford − Identification of hepatotoxic potential earlier (cost and time savings) − Opportunities for ranking and prioritizing or development of alternatives with lower toxicity Multiparameter high content cell-based screening methods in drug discovery contribute to better predictivity of human hepatotoxicity potential Early safety screening current priority in drug development
    • Early Safety Hepatotoxicity Screening Assays Development of effective in vitro cell-based screening models to assess human hepatotoxicity potential of drugs ideally requires: Use of high content multiplexed technologies Utilization of hepatocyte models (human and rat primary cells) Measurement of parameters − At the single cell level − Morphological and biochemical − Investigative of pre-lethal cytotoxic effects − Representative of different mechanisms of toxicity − Suitable for rapid throughput Minimal amount of compound for testing (1 - 2 mg)
    • MDS Multiplexed High Content Screening Tools: IN Cell 1000 Analyzer automated fluorescent microscopy imaging of live or fixed cells allows Subcellular localization AND quantitation of the cellular targets Multiplexing capabilities: multiple data points from a single assay well High sensitivity (nuclear staining allows for normalization of cellular signals against cell number) Measurement of individual cell responses in the heterogeneous cell populations Customized protocols for cell image quantitation (IN Cell Developer Software) xMAP technology using Luminex Flow based multiplexed microsphere assay system Multi-analyte protein analysis in the same well Nuclei staining with IN Cell imaging allows normalization of cellular signals against cell number IT Support XLFit Curve-fitting software/template (embedded macros for handling multiple plates) AIM automated Data Analysis and Report Generation
    • MDS in vitro Multiplexed High Content Screening Hepatotoxicity Early Safety Platform HCS Hepatotoxicity Early Safety Platform Hepato-toxicity (cell proliferation, apoptosis, mitosis) Hepato-Lipid Accumulation (cell proliferation, phospholipidosis, neutral lipids) Hepato-Cytokine Secretion (cell proliferation, inflammatory markers)
    • Non-contact dispensing automation system for compound addition, cell fixing and immunostaining Titertek Multidro p Titertek Multidrop ultidrop T itertek M asher V-Spin Centrifuge 05 w ELx4 LiCONiC CO2 Biote k incubator Labcyte® Echo™ 550 De-lidder Carousel w/ 12 hotels, 16 slots each (not shown) Velocity 11 BioCel® 1200 system
    • MDS AIM automated Data Analysis and Report Generation Includes curve fitting, data visualization, quality and statistical assessment Concentration Relative cell Apoptosis (fold Mitosis (fold (microM) count (%) induction) induction) Mean StdDev Mean StdDev Mean StdDev 3.18E-03 96.9 2.6 0.6 0.6 1.1 0.2 1.01E-02 92.9 5.8 1.7 0.6 1.4 0.1 3.18E-02 95.3 6.3 1.6 1.1 0.9 0.1 1.00E-01 93.5 3.6 1.2 0.6 0.9 0.0 3.17E-01 94.2 8.3 0.8 0.2 1.0 0.1 1.00E+00 100.2 12.5 1.0 0.8 0.9 0.0 3.17E+00 89.3 3.6 2.0 0.7 0.8 0.0 1.00E+01 25.2 1.3 4.1 0.8 0.6 0.1 3.16E+01 18.1 2.5 10.4 7.9 0.5 0.2 1.00E+02 7.9 1.5 67.0 8.9 0.6 0.1 Blue indicates that values meet the statically significant response criteria specified in the data interpretation chapter.
    • Multiplexed In vitro Hepatotoxicity Assay In vitro hepatotoxicity assessment Cultured HepG2 cells (human hepatocellular carcinoma cell line) useful screening reagent Evaluation of toxicity ‘window / safety margin’ and mechanism of death helps determine dosing and cost/benefit analysis of therapeutic agent based on prediction of in vivo toxicity potential − In vitro cell-based safety margin = cytotoxic concentration – on-target potency concentration (cell-based efficacy) − Higher values predict higher in vivo safety margins − In vitro cell-base safety margins use to rank compounds based on hepatotoxicity potential in humans − 80% correlation between actual in vivo and in vitro cell-based toxicity results have been demonstrated (Shrivastava R, et al., O’Brien PJ, et al., Vivek C, et al.) − Other factors contributing to toxicity profiles: drug properties, concentrations, protein binding and transport, pharmacokinetic characteristics Provides information on the relative toxicities of candidate drugs within particular compound families to aid selection of lead candidates. Offers insight into drug toxicity mechanism Provides end-point-specific drug hepatotoxicities
    • Multiplexed In vitro Hepatotoxicity Assay Multiplexed Hepatotoxicity Assay HepG2 cells seeded in 384-well Collagen I coated optical plates, incubated 24 hrs Cells incubated 72 hrs with test compounds serially diluted ½ log over 10 concentrations Post 72 hrs incubation cells fixed and immunolabeled with: − Anti-active Caspase-3 for detection of apoptosis − Anti-phospho-Histone-3 for detection of cell cycle − Stained with a nuclear dye for cell proliferation quantification Automated fluorescence microscopy carried out using a GE Healthcare IN Cell Analyzer 1000 Images collected with a 4X objective
    • Multiplexed In vitro Hepatotoxicity Assay Data Analysis Total (masked) fluorescent intensities instead of individual cell counts results in − Higher throughput (reading time savings: 4X, 20 min/plate vs 20X, 2 hrs/plate) − Lower CVs (more cells analyzed) For relative cell counts, percent of control (POC), a ratio of the fluorescent intensity of treated wells to intensity from the control wells, is used for normalization Nx/Nc = POC For activated Caspase-3 and phospho-Histone-3, each intensity is first normalized to the nuclear intensity in their prospective wells. Then this ratio of each well is normalized to the control wells (Cx/Nx) / (Cc/Nc) = Fold induction over vehicle Cx=Caspase intensity for well X Nx= Nuclear intensity for well X Cc=Caspase intensity for control well Nc=Nuclear intensity for control well
    • Advantages of Multiplex Hepatotoxicity Assay Data Output Relative cell number quantified by total nuclear Intensity Advantageous over cell count due to cells that were hard to mask (over lapping cells) Allowed use of 4X objective to capture more cells (less imaging time with better statistics) Used less image storage space Output: EC50 / IC50 − Relative cell count IC50 = test compound concentration that produces 50% of the cell proliferation inhibitory response or 50% cytotoxicity level − Relative cell count EC50 = test compound concentration that produces 50% of the maximum effective response, accounts for cells not killed (curve inflection point) Apoptosis: Measured by activated Caspase-3 antibody Detected a wide range of dying and dead cells (from early to late apoptosis) Robust signal Output: [ ] at 5-fold over background Cell Cycle: Measured by phospho-Histone-3 antibody Measure of cells in mitotic phase Could also detect buildup in G1/S by decrease in mitotic signal from that of control Output: [ ] at 2-fold over background or 2-fold below background
    • Multiplexed In vitro Hepatotoxicity Assay Vehicle Vinblastine Labels: Nuclei - green; Apoptotic cells - blue; Mitotic cells - red Cell Proliferation Apoptosis Induction Cell Cycle Block Percent of Control over Background over Background Fold Induction 160 100 6 Fold Induction 140 80 120 100 60 4 80 60 40 2 40 20 20 0 0 -13 -12 -11 -10 -9 -8 -7 -6 0 -13 -12 -11 -10 -9 -8 -7 -6 -13 -12 -11 -10 -9 -8 -7 -6 [Vinblastine], M [Vinblastine], M [Vinblastine], M
    • Multiplexed In Vitro Hepatotoxicity Assay Hepatotoxicity assay parameters for compounds tested, (n = 3) Inhibition of Apoptosis Mitosis mitosis (G1/S Relative cell Relative cell induction cell cycle block cell cycle count count [ ] at 5-fold Compound [ ] at 2-fold over block) [ ] at 2- IC50 EC50 over background fold below (microM) (microM) background (microM) background (microM) (microM) Propranolol 62.81 ± 5.23 61.38 ± 5.38 55.28 ± 5.43 − − Staurosporine 0.036 ± 0.005 0.027 ± 0.003 0.036 ± 0.005 − 0.144 ± 0.018 Cyclosporin A 7.71 ± 0.76 5.43 ± 0.30 9.99 ± 0.29 − − Vinblastine 0.002 ± 0.000 0.002 ± 0.000 0.003 ± 0.001 0.002 ± 0.000 − Erythromycin > 100 > 100 − − − All values are given as the mean ± s.e.m.
    • Multiplexed In vitro Hepato-Lipid Accumulation Assay In vitro hepato-lipid accumulation assessment − Cultured HepG2 cells (human hepatocellular carcinoma cell line) Phospholipidosis accumulation of excess phospholipids in cells − Cationic amphiphilic drugs often induce phospholipidosis in vivo − Toxic effect due to drug or metabolite accumulation in affected tissue, leads to acute and chronic disease − Liver and lung common targets Neutral lipid accumulation − Steatosis accumulation of fatty acids − Other mechanisms of lipid accumulation − Can cause enlargement of the liver and irreversible cell damage Flags drug candidate hepatotoxicity potential in the lead optimization stage of drug discovery End-point-specific drug-induced mechanism of hepatotoxicity
    • Multiplexed In vitro Hepato-Lipid Accumulation Assay Multiplexed Hepato-Lipid Accumulation Assay HepG2 cells seeded in 384-well Collagen I coated optical plates, incubated 24 hrs Cells incubated for 48 hrs with − Fluorescently-labeled phospholipid (Invitrogen, H34350) for phospholipid accumulation detection − Test compounds serially diluted ½ log over 10 concentrations Post 48 hrs incubation cells fixed and stained with − Neutral lipid dye (Invitrogen, H34476) for neutral lipid detection − Nuclear dye for cell proliferation quantification Automated fluorescence microscopy carried out using a GE Healthcare INCell Analyzer 1000 Images were collected with a 4X objective.
    • Multiplexed In vitro Hepato-Lipid Accumulation Assay Hepato-Phospholipid Accumulation assay Labels: Nuclei - green; Phospholipids - red
    • Multiplexed In vitro Hepato-Lipid Accumulation Assay
    • Multiplexed In vitro Hepato-Lipid Accumulation Assay Hepato-Neutral Lipid Accumulation Assay Labels: Nuclei - green; Neutral lipids - red
    • Advantages of Multiplexed In vitro Hepato- Lipid Accumulation Assay Data Output Relative cell number quantified by total nuclear Intensity Advantageous over cell count due to cells that were hard to mask (over lapping cells) Allowed use of 4X objective to capture more cells (less imaging time with better statistics) Used less image storage space Output: EC50 / IC50 − Relative cell count IC50 = test compound concentration that produces 50% of the cell proliferation inhibitory response or 50% cytotoxicity level − Relative cell count EC50 = test compound concentration that produces 50% of the maximum effective response, accounts for cells not killed (curve inflection point) Phospholipidosis: Measured by fluorescently labeled phospholipid Detected phospholipid accumulation Robust signal Output: [ ] at 5-fold over background Neutral lipids: Measured by neutral lipid dye Detected neutral lipid accumulation (steatosis/cholestasis) Robust signal Output: [ ] at 5-fold over background
    • Multiplexed In vitro Hepato-Lipid Accumulation Assay Hepato-lipid accumulation assay parameters for each compound tested, (n = 3)
    • Multiplexed In vitro Hepato-Cytokine Secretion Assay Multiplexed Hepato-Cytokine Secretion Assay IN Cell xMAP™ Automated technology fluorescent using microscopy Luminex imaging Markers of cell count inflammation normalization
    • xMAP technology-Multiple Analytes/Well Multiplexing: Up to 100 analytes/well Analytes cytokines or other inflammatory markers Flow based assay system. Uses beads loaded with different concentrations of 2 dyes. Each bead has it’s own unique spectral signature (100 possible), antibodies are derivitized to unique bead Beads are incubated with test sample Sandwich assay performed with a biotinylated second antibody (mouse) Streptavidin labeled with phycoerythrin (PE) used for detection Beads are run individually (Flow) through a laser which detects the exact bead and then determines whether PE is associated
    • Multiplexed In vitro Hepato-Cytokine Secretion Assay Multiplexed Hepato-Cytokine Secretion Assay Biomarker secretion, as markers of inflammation Nuclear count, analyte normalization to cell number HepG2 cells seeded into 96-well Collagen I coated optical plates incubated 24 hrs Cells treated with LPS, TNFα, IL-1β and acetaminophen serially diluted ½ log over 8 concentrations incubated 48 hrs Post 48 hrs incubation supernatants collected, cytokine detection was carried out using Luminex xMAP™ technology To quantify cell proliferation the monolayer of HepG2 cells remaining in each plate was immediately stained with nuclear dye for normalization Images were collected using a GE Healthcare INCell Analyzer 1000
    • Multiplexed In vitro Hepato-Cytokine Secretion Assay HepG2 cells treated with LPS, TNFα, IL-1β and acetaminophen HepG2 cells Screened for the secretory presence of 30 human inflammatory markers: LPS, TNFα, IL-1β and acetaminophen IL-1α, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p40, IL- 12-70, IL-13, INFγ, INFα2a, Fibrinogen, Apo AI, Apo AII, Apo B, IP-10, GM-CSF, G-CSF, CRP, Haptoglobin, Apo CII, Apo CIII and MCP-1, MIP-1α, MIP-1β, SAA Apo E TNFα, IL-1 receptor antagonist
    • Advantages of Multiplexed In vitro Hepato- Cytokine Secretion Assay Data Output Relative cell number quantified by total nuclear Intensity Advantageous over cell count due to cells that were hard to mask (over lapping cells) Allowed use of 4X objective to capture more cells (less imaging time with better statistics) Used less image storage space Output: percent of control (POC) − Relative cell count POC, ratio of the fluorescent intensity in treated wells to intensity from control wells, used for normalization POC = Nx/Nc Nx= Nuclear intensity for well X Nc=Nuclear intensity for control well Inflammatory marker secretion: multiplexed Luminex xMAP™ technology Detected biomarker secretion Normalized by nuclear intensity POC as a measure of relative cell count Robust signal Output: − [ ] of biomarker secretion at 3-fold over background − Emax (maximum [ ] of secreted biomarker)
    • Multiplexed In vitro Hepato-Cytokine Secretion Assay
    • Early Safety Screening for Mechanisms of Hepatotoxicity Conclusion: We have developed a robust and rapid throughput screening system using HepG2 cells that allows early assessment of acute and chronic mechanisms of hepatotoxicity Compounds with known hepatotoxicities tested in validating the capabilities of this multiparametric HCS system in identifying and quantifying toxicities relevant to cell proliferation, apoptosis, cell cycle, steatosis/cholestasis and phospholipidosis demonstrated high concordance with reported hepatotoxic profile for each compound tested Evaluation of cytokine secretion in HepG2 cells to identify measurable biomarkers of inflammation demonstrated significant secretion levels for 6 of the cytokines tested thus validating this multiplexed approach for quantifying indications of hepatic inflammation These hepatotoxicity screening assays are sensitive and reproducible and provide results that previously only have been attainable in more complex in vivo models Our cost-effective in vitro multiplexed HCS platform offers comprehensive predictive information allowing pre-selection of drug scaffold designs with long-term hepatotoxicity considerations and may even have more relevance when performed in normal primary hepatocytes
    • Acknowledgements Cell Biology Team Automation Christine O’Day Robert Keyser Yulia Ovechkina Phuong TB Nguyen IT Rod Shively Mike Harges Jenny Mulligan Mark Taylor Cheryl Bogucki Ed Gonterman
    • Automated compound addition using non-contact acoustic based system, Labcyte® Echo™ 550 Inverted receiving plate with cells Source plate with compound in DMSO solution Piezoelectric transducer (a). Piezoelectric (b). Compound in (c). When the cell transducer produces DMSO sitting at assay plate is inverted focused acoustic the meniscus of back to its up-right waves to transfer cell media after position, compound in compound. transfer. DMSO diffuses toward the cell monolayer. One well of 384 well plate is shown.
    • Customized cell image quantitation protocols are constructed with IN Cell Developer software Wide range of quantitative measures Flexible, multifunctional and user- friendly protocol editor Fast analysis Batch capabilities